Sabine Hüwel

Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier.

The blood-brain barrier (BBB) plays the predominant role in controlling the passage of endogenous and xenobiotic substances between the circulating blood and the extracellular fluid environment of the brain. The transfer of compounds is strictly regulated by brain capillary endothelial cells (BCEC), which are interconnected to each other by well developed tight junctions, without fenestrations. Although hydrophobic molecules such as nicotine and ethanol readily cross the BBB by diffusion, the brain microvasculature shows a highly restrictive permeability to hydrophobic antitumor agents. So far, this multidrug resistance has been almost exclusively attributed to the most prominent member of the ATP-binding cassette (ABC) transporter family, P-glycoprotein located in the luminal membrane of brain capillary endothelial cells and to a minor extent to the multidrug resistance-associated proteins (MRPs). The brain multidrug resistance protein (BMDP) has recently been discovered at the porcine BBB and was shown to be highly homologous to the human breast cancer resistance protein (BCRP/ABCG2). Here, we demonstrate by northern blot and RT-PCR analysis that BMDP mRNA is more highly expressed in the capillary endothelial cells compared to other cell types of the brain. Immunocytochemistry of porcine BCEC showed a clear plasma membrane localisation of BMDP. Analysis of the total mRNA pool revealed that BMDP is more strongly expressed than P-glycoprotein and MRP1. Consistently, first transport studies indicate that active exclusion of the chemotherapeutic drug daunorubicin from the central nervous system is mediated mainly by this new transporter compared to P-glycoprotein or MRP1. Thus, we hypothesise that BMDP might play an important role in the exclusion of xenobiotics from the porcine brain.

Receptor-mediated delivery of magnetic nanoparticles across the blood-brain barrier.

A brain delivery probe was prepared by covalently conjugating lactoferrin (Lf) to a poly(ethylene glycol) (PEG)-coated Fe(3)O(4) nanoparticle in order to facilitate the transport of the nanoparticles across the blood-brain barrier (BBB) by receptor-mediated transcytosis via the Lf receptor present on cerebral endothelial cells. The efficacy of the Fe(3)O(4)-Lf conjugate to cross the BBB was evaluated in vitro using a cell culture model for the blood-brain barrier as well as in vivo in SD rats. For an in vitro experiment, a well-established porcine BBB model was used based on the primary culture of cerebral capillary endothelial cells grown on filter supports, thus allowing one to follow the transfer of nanoparticles from the apical (blood) to the basolateral (brain) side. For in vivo experiments, SD rats were used as animal model to detect the passage of the nanoparticles through the BBB by MRI techniques. The results of both in vitro and in vivo experiments revealed that the Fe(3)O(4)-Lf probe exhibited an enhanced ability to cross the BBB in comparison to the PEG-coated Fe(3)O(4) nanoparticles and further suggested that the Lf-receptor-mediated transcytosis was an effective measure for delivering the nanoparticles across the BBB.

Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma

Loss of folate receptor-α function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-α-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-α, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-α-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-α-positive exosomes. Loss of folate receptor-α-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-α-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-α-positive and -negative exosomes into mouse brains demonstrate folate receptor-α-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-α-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting.

Transport of Poly(n-butylcyano-acrylate) nanoparticles across the blood-brain barrier in vitro and their influence on barrier integrity.

In previous studies it was shown that polysorbate 80(PS80)-coated poly(n-butylcyano-acrylate) nanoparticles (PBCA-NP) are able to cross the blood-brain barrier (BBB) in vitro and in vivo. In order to explore and extend the potential applications of PBCA-NP as drug carriers, it is important to ascertain their effect on the BBB. The objective of the present study was to determine the effect of PS80-coated PBCA-NP on the BBB integrity of a porcine in vitro model. This has been investigated by monitoring the development of the transendothelial electrical resistance (TEER) after the addition of PBCA-NP employing impedance spectroscopy. Additionally, the integrity of the BBB in vitro was verified by measuring the passage of the reference substances (14)C-sucrose and FITC-BSA after addition of PBCA-NP. In this study we will show that the application of PS80-coated PBCA-NP leads to a reversible disruption of the barrier after 4h. The observed disruption of the barrier could also be confirmed by (14)C-sucrose and FITC-BSA permeability studies. Comparing the TEER and permeability studies the lowest resistances and maximal values for permeabilities were both observed after 4h. These results indicate that PS80-coated PBCA-NP might be suitable for the use as drug carriers. The reversible disruption also offers the possibility to use these particles as specific opener of the BBB. Instead of incorporating the therapeutic agents into the NP, the drugs may cross the BBB after being applied simultaneously with the PBCA-NP.

Impact of Manganese on and Transfer across Blood-Brain and Blood-Cerebrospinal Fluid Barrier in Vitro

Background: Modes of neurotoxic action after Mn overexposure are not fully understood. Results: The blood-CSF barrier showed higher Mn sensitivity and active Mn transport properties. Conclusion: The blood-CSF barrier might be the major route for Mn into the brain. Significance: Deeper insight in the impact of Mn on and its transfer across brain barrier systems might help to prevent irreversible neurological damage. Manganese occupational and dietary overexposure has been shown to result in specific clinical central nervous system syndromes, which are similar to those observed in Parkinson disease. To date, modes of neurotoxic action of Mn are still to be elucidated but are thought to be strongly related to Mn accumulation in brain and oxidative stress. However, the pathway and the exact process of Mn uptake in the brain are yet not fully understood. Here, two well characterized primary porcine in vitro models of the blood-brain and the blood-cerebrospinal fluid (CSF) barrier were applied to assess the transfer of Mn in the brain while monitoring its effect on the barrier properties. Thus, for the first time effects of MnCl2 on the integrity of these two barriers as well as Mn transfer across the respective barriers are compared in one study. The data reveal a stronger Mn sensitivity of the in vitro blood-CSF barrier compared with the blood-brain barrier. Very interestingly, the negative effects of Mn on the structural and functional properties of the highly Mn-sensitive blood-CSF barrier were partly reversible after incubation with calcium. In summary, both the observed stronger Mn sensitivity of the in vitro blood-CSF barrier and the observed site-directed, most probably active, Mn transport toward the brain facing compartment, reveal that, in contrast to the general assumption in literature, after oral Mn intake the blood-CSF barrier might be the major route for Mn into the brain.

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.

How does the benzamide antipsychotic amisulpride get into the brain?--An in vitro approach comparing amisulpride with clozapine.

This study evaluated the disposition of the two atypical antipsychotics, amisulpride (AMS) and clozapine (CLZ), and its main metabolite N-desmethylclozapine (DCLZ), to their target structures in the central nervous system by applying an in vitro blood–brain barrier and blood–cerebrospinal fluid (CSF) barrier based on monolayers of porcine brain microvessel endothelial cells (PMEC) or porcine choroid plexus epithelial cells (PCEC). Permeation studies through PMEC- and PCEC-monolayers were conducted for 60 min at drug concentrations of 1, 5, 10, and 30 μM applied to the donor compartment. PMEC were almost impermeable for AMS (permeation coefficient, P<1 × 10−7 cm/s) in the resorptive direction, whereas transport in the secretory direction was observed with a P (±SD) of 5.2±3.6 × 10−6 cm/s. The resorptive P of CLZ and DCLZ were 2.3±1.2 × 10−4 and 9.6±5.0 × 10−5 cm/s, respectively. For the permeation across PCEC in the resorptive direction, a P of 1.7±2.5 × 10−6 cm/s was found for AMS and a P of 1.6±0.9 × 10−4 and 2.3±1.3 × 10−5 cm/s was calculated for CLZ and DCLZ, respectively. Both, CLZ and DCLZ, could easily pass both barriers with about a five-fold higher permeation rate of CLZ at the PCEC. The permeation of AMS across the BBB was restricted partly due to an efflux transport. It is thus suggested that AMS reaches its target structures via transport across the blood–CSF barrier.

Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood–brain barrier

Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

Permeability of ergot alkaloids across the blood‐brain barrier in vitro and influence on the barrier integrity

Scope Ergot alkaloids are secondary metabolites of Claviceps spp. and they have been in the focus of research for many years. Experiments focusing on ergotamine as a former migraine drug referring to the ability to reach the brain revealed controversial results. The question to which extent ergot alkaloids are able to cross the blood-brain barrier is still not answered. Methods and results In order to answer this question we have studied the ability of ergot alkaloids to penetrate the blood-brain barrier in a well established in vitro model system using primary porcine brain endothelial cells. It could clearly be demonstrated that ergot alkaloids are able to cross the blood-brain barrier in high quantities in only a few hours. We could further identify an active transport for ergometrine as a substrate for the BCRP/ABCG2 transporter. Investigations concerning barrier integrity properties have identified ergocristinine as a potent substance to accumulate in these cells ultimately leading to a weakened barrier function. Conclusion For the first time we could show that the so far as biologically inactive described 8-(S) isomers of ergot alkaloids seem to have an influence on barrier integrity underlining the necessity for a risk assessment of ergot alkaloids in food and feed.

In vitro intestinal bioavailability of arsenosugar metabolites and presystemic metabolism of thio-dimethylarsinic acid in Caco-2 cells

Two arsenosugar metabolites show in vitro intestinal bioavailability similar to that of arsenite, and much higher than that of arsenosugars.